Abstract: 【Objective】 Phloridzin oxidation product 2 (POP2) is a final polyphenol oxidation product generated by enzymatic oxidation in the process of fruit juice, and it has an important impact on the browning and deterioration of fruit juice. In order to detect POP2 effects during process and storage of apple juice, the phloridzin polyphenol oxidase reaction system was established, POP2 was separated and purified, structural characterization was analyzed. 【Method】 The three reaction factors of pH, temperature and substrate concentration were tested first, and then enzyme reaction in ethanol solution were studied, and content changes of phloridzin, intermediate products, POP2 during oxidation process were detected by HPLC method. The POP2 was isolated and purified by ethyl acetate extraction, anhydrous ethanol desalting and recrystallization, the structure of POP2 was characterized by the methods of ESI-MS and IR.【Result】 The optimal conditions for the enzymatic oxidation of phloridzin were pH 6.5, 35℃, substrate concentration 3 mg?mL^-1. The addition of different concentrations of absolute ethanol had a significant increase in the rate of enzymatic oxidation of phloridzin, the highest reaction conversion rate was 2.4 times at 20% ethanol concentration. The color of the phloridzin reaction fluid changes from slightly yellow, bright yellow, orange to orange-red at the beginning, middle and final reaction stage. The HPLC results showed that the conversion rate of phlorizin was 90.2% when the substrate concentration was 3 mg?mL-1 and the reaction time was 48 h. Intermediates X1 and POP1 were almost close to trace, the accumulated POP2 was stable. The residual phloridzin in the reaction solution could be removed by ethyl acetate extraction. The purity of 96.8% POP2 was obtained by anhydrous ethanol desalting and recrystallization from methanol. The molecular weight of POP2 was 467 by positive mode ESI-MS analysis, molecular formula is C₂₁H₂₂O₁₂. IR results showed that POP2 had characteristic ether, ketone, benzene ring and carboxyl functional groups, and it was consistent with the structure of the predicted phlorizin oxidation product structure. 【Conclusion】 The major oxidation products of phloridzin in enzymatic system is POP2, POP2 content can be established a landmark test indicators in apple juice browning and deterioration.

Key words: fruit juice, phloridzin, oxidation products POP2, enzymatic reaction, structure characterization